Biological production of organic compounds

ABSTRACT

Strains of cyanobacteria that produce high levels of alpha ketoglutarate (AKG) and pyruvate are disclosed herein. Methods of culturing these cyanobacteria to produce AKG or pyruvate and recover AKG or pyruvate from the culture are also described herein. Nucleic acid sequences encoding polypeptides that function as ethylene-forming enzymes and their use in the production of ethylene are further disclosed herein. These nucleic acids may be expressed in hosts such as cyanobacteria, which in turn may be cultured to produce ethylene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/512,075, filed Jul. 27, 2011, the contents of which are incorporatedby reference in their entirety.

CONTRACTUAL ORIGIN

The United States Government has rights in this invention under ContractNo. DE-AC36-08G028308 between the United States Department of Energy andthe Alliance for Sustainable Energy, LLC, the Manager and Operator ofthe National Renewable Energy Laboratory.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file entitled “11-18_ST25.txt,” having a size in bytes of 20 kb andcreated on Jul. 27, 2012. Pursuant to 37 CFR §1.52(e)(5), theinformation contained in the above electronic file is herebyincorporated by reference in its entirety.

BACKGROUND

Using algae or cyanobacteria to produce carbon compoundsphotosynthetically from CO₂ and water has theoretical potential but hasyet to be realized on an industrial scale. One major limitation is thatthese cells are naturally high in protein, especially under conditionsthat lead to maximum growth rates. Thus a large fraction of carbon fromphotosynthesis is used to produce nitrogen containing amino acids ratherthan other products containing only carbon, hydrogen, and oxygen. Whennitrogen sources are removed from cultures of non-diazotrophiccyanobacteria, cells accumulate high concentrations of glycogen, butlarge-scale harvesting of microbial oxygenic phototrophs is difficultand expensive with currently available technologies.

Alpha ketoglutarate (AKG) is used as an organic synthesis intermediate,a medicine ingredient, a biochemical reagent, and as a nutritionaladditive in food and sport drinks. Currently AKG is produced by chemicalsynthesis using triethyl oxalosuccinic ester derived from petroleum andconcentrated hydrochloric acid, or by fermentation using sugar asfeedstock. Photosynthetic production of AKG could therefore replacepetroleum or sugar as the feedstock and eliminate the use of corrosiveacid.

Ethylene is used in the synthesis of diverse products from plastics(e.g., polyethylene, polystyrene, and PVC) to textiles such aspolyester. Ethylene has been used to produce high-grade ethanolindustrially for the past 50 years, by a relatively simple catalyticprocess involving the hydration of ethylene into ethanol. In addition,the technology to polymerize ethylene to gasoline has been known fornearly a century. Ethylene is the most widely produced organic compoundglobally, with more than 132.9 million tons produced in 2010 andprojected growth of 5% a year through 2015.

The current method of producing ethylene is via steam cracking of longchain hydrocarbons from petroleum, or via dehydrogenation of ethane.Unfortunately, fossil fuel supplies are finite and utilization of thesefeed stocks produces greenhouse gases such as CO₂ (1.5 to 3.0 tons CO₂per ton of ethylene). For these reasons, sustainable, carbon neutralprocesses that are capable of producing this essential chemical areneeded. One such alternative is the use of biological processes toconvert CO₂ or other waste products into ethylene. Based on the overallequation 2CO₂+2H₂O═C₂H₄+3O₂, photosynthetic production of one ton ofethylene could sequester 3.14 tons of CO₂.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods that aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

Exemplary embodiments provide methods for producing alpha ketoglutarate(AKG) or pyruvate by culturing a cyanobacterial cell that lacks afunctional ADP-glucose pyrophosphorylase (AGP) enzyme under conditionsthat allow for AKG or pyruvate production and recovering the AKG orpyruvate from the cyanobacterial cell culture.

In some embodiments, the cyanobacteria) cell does not express afunctional glgC gene and/or is a Synechocystis cell such as aSynechocystis sp. PCC 6803 cell.

In certain embodiments, the cyanobacterial cell is cultured undernitrogen starvation conditions in media that does not contain nitrogen.In some embodiments, the concentration of nitrogen in the media is lessthan about 200 μM. The methods may include a step of adding nitrogen tothe media at a final concentration of less than about 1 mM. In certainembodiments, the cyanobacterial cell is cultured under a light intensityof at least about 350 or 600 μE m⁻²s⁻¹.

In further embodiments, the AKG concentration in the culture is greaterthan 100 mg per liter or 1000 mg per liter. In some embodiment, thepyruvate concentration in the culture is greater than 1 g per liter or100 g per liter. In certain embodiments, the cyanobacterial cellexhibits at least a 10,000-fold increase in AKG and/or pyruvateproduction when compared to the wild type cell.

Also provided are cyanobacterial cells that lack a functionalADP-glucose pyrophosphorylase (AGP) enzyme and produce 10,000-fold moreAKG or pyruvate when compared to a wild type cell. In some embodiments,the cyanobacterial cells do not express a functional glgC gene and/orare Synechocystis cells such as a Synechocystis sp. PCC 6803 cells.

Further provided are isolated nucleic acid molecules with sequences atleast 90% identical to SEQ ID NO:3 that encode polypeptides thatfunction as ethylene-forming enzymes. In some embodiments, the nucleicacid molecule has a sequence at least 95% identical to SEQ ID NO:3 orhas or comprises the sequence of SEQ ID NO:3.

In certain embodiments, the nucleic acid molecule further comprises apromoter such as petE or psbA operably linked to the nucleic acidmolecule.

Also provided are expression vectors comprising nucleic acid moleculeswith sequences at least 90% identical to SEQ ID NO:3 that encodepolypeptides that function as ethylene-forming enzymes.

Exemplary embodiments also provide host cells comprising expressionvectors described herein or expressing recombinant polypeptides encodedby the nucleic acids described herein. In some embodiments, the hostcell is a microbial cell, a cyanobacterial cell, a Synechocystis cell ora Synechocystis sp. PCC6803 cell. In certain embodiments, the cellmaintains a functional copy of the ethylene-forming enzyme for at leastfour generations. In certain embodiments, the host cells comprise atleast one, two, three, four, five or more copies of efe.

Additional embodiments provide methods for producing ethylene comprisingculturing a host cell that expresses a recombinant ethylene-formingenzyme under conditions that allow for ethylene production and isolatingethylene from the culture.

In some embodiments, the host cell is a Synechocystis cell and expressesa nucleic acid molecule with a sequence at least 90% identical to SEQ IDNO:3.

In certain embodiments, the method further comprises a step ofreplenishing components of the culture medium that are depleted duringthe culturing step to the cell culture.

In exemplary embodiments, ethylene is produced at a peak production rateof at least 500 nL mL⁻¹ hr⁻¹, at least 1 μL mL⁻¹ hr⁻¹, at least 10 μLmL⁻¹ hr⁻¹, at least 50 μL mL⁻¹ hr⁻¹, at least 100 μL mL⁻¹ hr⁻¹, or atleast 200 μL mL⁻¹ hr⁻¹.

In some embodiments, the step of culturing comprises providing the cellcarbon dioxide and light. The light may be sunlight and the carbondioxide may be atmospheric carbon dioxide.

Further provided are methods for producing ethylene comprising culturinghost cells as described herein under conditions that allow for theproduction of ethylene by the cells and isolating the ethylene producedby the cells.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than limiting.

FIG. 1 illustrates the major carbon biosynthetic pathways incyanobacteria such as Synechocystis 6803 under nitrogen-deprivedconditions.

FIG. 2 shows growth curves of batch cultures of Synechocystis sp. PCC6803 cultured in full BG11 media (A) and after suspension in BG11-Nmedia (B). Cultures were under 24 hour light at ˜50 μEm⁻²s⁻¹ light flux.Panel (C) shows growth curves for wild type and mutant strains undernitrogen deprivation conditions.

FIG. 3 shows glycogen content of cultures from log phase (growing inBG11) and after incubation in BG11-N for 3 days. No glycogen wasdetected for the AGP-strain in either condition. Error bars representstandard deviation of biological replicates (n=3).

FIG. 4 illustrates light-adapted quantum efficiency (Fv′/Fm′) of cellscultured in BG11-N media for up to 5 days. Wild type (WT) is shown asdashed line because of phycobilin changes that artificially increase themeasured value. Error bars represent standard deviation of biologicalreplicates (n=3).

FIG. 5 shows whole-chain net oxygen evolution of cells cultured inBG11-N media normalized to the volume of culture measured (mL) withrespect to days nitrogen-starved. Oxygen evolution rates are shown as apercentage of non-nitrogen starved cells. Non-nitrogen starved WT andmutant (AGP-) cultures were within statistical error of each other (˜150μmmol O₂*(mg Chla)⁻¹*hr⁻¹). Error bars represent standard deviation ofbiological replicates (n=3).

FIG. 6 shows relative intracellular concentrations of select metaboliteswith and without nitrogen starvation (+N or −N) as determined by GC/MS.Y-axis is in units of “normalized ion count,” which accounts for thesignal response of a 0.1 mg/mL derivatized standard and the dry weightof the sample. Error bars represent standard deviation of biologicalreplicates (n=3).

FIG. 7A shows AKG production over 11 days of culturing in BG11-N mediaat a cell density of about 0.5 g/L cell dry weight and light flux ofabout 50 μEm⁻²s⁻¹. FIG. 7B shows pyruvate production over 5 days underthe same conditions. Error bars represent standard deviation ofbiological replicates (n=3).

FIG. 8 shows total dry weight of photosynthetically produced materialover 11 days of nitrogen starvation. Wild-type (WT) cells producednearly undetectable levels of excreted AKG, while a significant amountof AKG was produced by the AGP-mutant strain and is stacked over totalcell dry weight to show total dry weight material produced.

FIG. 9 shows dry weights of WT and AGP-cultures incubated for up to 9days in BG11-N.

FIG. 10 illustrates absorbance of whole cell suspensions at 630 nmdivided by absorbance at 680 nm, used to estimate phycobilin(PC)/Chlorophyll ratio in cells with respect to days of incubation inBG11-N.

FIG. 11 shows the nucleic acid sequence for the glgC gene (A; SEQ IDNO:1) and the amino acid sequence for the AGP protein (B; SEQ ID NO:2).

FIG. 12 illustrates the overall scheme for glgC gene replacement (A) andthe assembly of the glgC (shown here as agp) replacement construct usingfusion PCR.

FIG. 13 shows a PCR analysis illustrating that the glgC genomic fragmentis only detectable in the wild type genome.

FIG. 14 illustrates a pathway for the synthesis of ethylene via anethylene forming enzyme (EFE) that converts α-ketoglutarate, a keymetabolite in the citric acid cycle, to ethylene.

FIG. 15 shows growth rates of threes strains of Synechocystis 6803.

FIG. 16 shows ethylene production from Synechocystis 6803 harboring anefe gene. A) Comparison of ethylene production rates when efe isexpressed from either the petE or the psbA promoters. B) Ethyleneproduction normalized to density and total ethylene production (C) ofSynechocystis 6803 expressing efe from the psbA promoter across fourconsecutive cultures.

FIG. 17 illustrates growth conditions affecting ethylene production. A)Restoration of ethylene production by various additions to a stationaryculture that has ceased high-level ethylene production. B) The effect ofvarious medium concentrations on the total ethylene production. C)Refreshing the medium provides a higher rate of ethylene productionwhile allowing the peak production rates to be sustained. Arrowsindicate times at which the culture was resuspended in fresh medium.Free squares represent the ethylene production rates of the same culturebefore resuspension in fresh medium.

FIG. 18 shows a comparison of ethylene production as a function of time(A) or peak production rates (B) from strains harboring one, two, three,or four copies of efe in their genomes. In FIG. 18A, closed circlesrepresent one copy of efe, closed triangles represent two copies of efe,and the wild type strain is represented by the open circles.

FIG. 19 shows ethylene total productivity (A), specific productivity (B)and growth (C) after resuspension of cultures in fresh 5×BG11 media overmultiple weeks. One copy of efe is represented by solid symbols, twocopies of efe by open symbols. Panel (D) shows resuspension of a strainharboring two copies of efe in fresh 5×BG11 daily to an OD₇₃₀ of 15.0.

FIG. 20 shows ethylene production under various light conditions. A)Total ethylene productivity from cultures at various light intensities.B) Specific ethylene productivity from cultures at various lightintensities.

FIG. 21 shows ethylene total productivity (A), specific productivity (B)and growth (C) under high and low light intensities. A strain with twocopies of efe grown at 600 μE m⁻²s⁻¹ is represented by the closedcircles, a strain with one copy of efe grown at 600 μE m⁻²s⁻¹ by theopen circles, a strain with two copies of efe grown at 50 μE m⁻²s⁻¹ bythe open triangles, and a strain with one copy of efe grown at 50 μEm⁻²s⁻¹ by the closed triangles.

FIG. 22 shows the DNA sequence of the efe gene modified for expressionin Synechoeystis 6803 (SEQ ID NO:3). The ATG start codon is illustratedin bold.

FIG. 23 shows the amino acid sequence encoded by the P syringae efe gene(SEQ ID NO:4).

FIG. 24 shows specific ethylene production of a strain harboring asingle copy of efe in different types of media. Closed circles—5×BG11;open circle—5×BG11 dissolved in seawater; closed triangle—seawatersupplemented with 4 mg/L phosphate and 150 mg/L nitrate; opentriangle—sea water (n=3).

DETAILED DESCRIPTION

Disclosed herein are strains of cyanobacteria that produce high levelsof alpha ketoglutarate (AKG) and pyruvate. One example is a mutantstrain of Synechocystis lacking the glgC gene (FIG. 12A), which does notproduce a functional AGP protein (FIG. 12B) or glycogen in detectablelevels but over-produces AKG and pyruvate. Methods of culturing thesecyanobacteria to produce AKG and pyruvate are also described herein.

Syneehocystis, a non-diazotrophic cyanobacterium, accumulates largeamounts of glycogen when starved of nitrogen. While not wishing to bebound to any particular theory, it is believed that carbon flux fromphotosynthesis can be rerouted away from a storage product (e.g.,glycogen) and toward an excreted product (e.g., AKG or pyruvate) bycreating stable mutational strains of cyanobacterial cells that areincapable of synthesizing storage products such as glycogen orglucosylglycerol. Such strains include those with disruptions ordeletions in the gene that encodes an ADP-glucose pyrophosphorylase(AGP), which catalyzes the conversion of alpha-d-glucose-1-phosphate toadenosine diphosphoglucose (ADP-glucose), the immediate precursor toglycogen. Such AGP-deficient (AGP-) strains may not make detectableamounts of glycogen under nitrogen starvation, but continue to fixcarbon photosynthetically at a rate similar to wild-type cells. Yieldsof AKG and pyruvate in such mutant strains may exceed 100% of theinitial cell dry weight of cultures incubated under continuous light ina nitrogen-free growth medium. In Synechocystis sp. PCC 6803, a glgCgene (slr1176) encodes an AGP enzyme.

Suitable AGP-strains include those with a genetic mutation to interruptexpression of glgC yet maintain the ability to grow and producemetabolites such as AKG or pyruvate under nitrogen starvationconditions. Additional mutations or deletions in genes in the glycogensynthesis pathway (e.g., glycogen synthase) may also result in theincreased production of AKG or pyruvate. Under nitrogen depletionconditions, the mutant strains may produce high levels of AKG, up to orgreater than 30% of the cell dry weight, which is on the order of a10,000-fold increase over the wild type. In certain embodiments, themutant strains may exhibit at least a 100, 200, 300, 400, 500, 1000,2500, 5000, 7500, or 10,000-fold increase in AKG production whencompared to the wild type strain. In some embodiments, the mutantstrains may exhibit at least a 100, 200, 300, 400, 500, 1000, 2500,5000, 7500, or 10,000-fold increase in pyruvate production when comparedto the wild type strain.

The photosynthetic AKG production rate may reach at least 150 grams perday per 1000 liter reactor at a cell density of 1 gram dry weight perliter. Pyruvate can be produced at a rate of at least 275 grams per dayper 1000 liter reactor at a cell density of 1 gram dry weight per liter.In some embodiments, AKG or pyruvate production rates may reach at leastabout 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300grams per day per 1000 liter reactor at a cell density of 1 gram dryweight per liter. AKG or pyruvate may be harvested from the growthmedium without the need to harvest and break cells, thus allowing acontinuous “milking” (verses batch culture) process.

AGP-strains may be used for photosynthetic AKG or pyruvate productionfrom only net substrates of CO₂, water, and sunlight. A continuous AKGor pyruvate production system in which the culture serves assolar-driven catalyst to convert CO₂ to AKG or pyruvate is alsocontemplated. As there would be no need to harvest cells in such asystem, AKG or pyruvate can be harvested continuously or in intervalsfrom the medium. The removal of AKG or pyruvate from the medium may helpkeep intracellular AKG or pyruvate concentration lower, to preventpossible feedback inhibition, and enhance the rate of production.

The nucleic acid sequence of the glgC gene (SEQ ID NO:1) and the aminoacid sequence of the glgC gene product (SEQ ID NO:2) are shown in FIGS.11A and B, respectively. Exemplary methods for disrupting the glgC geneby fusion PCR are provided in the Examples and figures, but any methodsuitable for disrupting, ablating or mutating genes in cells may beused. In certain embodiments, all or a portion of the targeted gene isreplaced with a selectable marker. Markers may be an inducible ornon-inducible gene and will generally allow for positive selection.Non-limiting examples of selectable markers include the ampicillinresistance marker (i.e., beta-lactamase), tetracycline resistancemarker, neomycin/kanamycin resistance marker (i.e., neomycinphosphotransferase), dihydrofolate reductase, glutamine synthetase, andthe like. The choice of the proper selectable marker will depend on thehost cell, and appropriate markers for different hosts as understood bythose of skill in the art.

Nucleic acid sequences encoding polypeptides that function asethylene-forming enzymes and their use in the production of ethylene arealso disclosed herein. These nucleic acids may be expressed in hostssuch as cyanobacteria, which in turn may be cultured to produceethylene. For example, methods for using the unicellular cyanobacteriumSynechocystis sp. PCC 6803 to photosynthetically produce ethylene fromatmospheric CO₂ are disclosed.

One function of ethylene is as a plant hormone that is involved inregulating numerous processes such as germination, senescence and fruitripening. In plants, ethylene is synthesized from methionine, which isfirst converted to S-adenosyl-L-methionine,L-aminocyclopropane-L-carboxylic acid, and finally to ethylene in athree step reaction. Some microbes are also capable of producingethylene through two pathways not found in plants. Most bacteriagenerate ethylene from methionine in a two step reaction with a2-keto-4-methyl-thiobutyric acid intermediate. This reaction, catalyzedby a NADH:Fe(III)EDTA oxidoreductase, is rather inefficient. Many plantpathogens such as Pseudomonas syringae synthesize ethylene duringinfection to weaken their host. In P. syringae, ethylene is synthesizedfrom the TCA cycle intermediate, alpha-ketoglutarate, in an efficientsingle step reaction catalyzed by the ethylene forming enzyme (EFE).

Atmospheric CO₂ represents a feedstock that has potential for conversioninto other chemicals such as ethanol, biodiesel, or ethylene usingphotosynthesis. To this end, researchers have attempted to use thisprocess, coupled with EFE, to generate ethylene from atmospheric CO₂.Previous attempts have failed due to poor productivity and the inabilityto stably express EFE (see Fukuda et al., Biotechnol. Lett. 16:1-6(1994); Sakai et al., J. Ferment. Bioeng. 84:434-443 (1997); Takahama etal., J. Biosci. Bioeng. 95:302-305 (2003)).

Disclosed herein are methods for expressing or overexpressing efe genes(for example, from P. syringe) in hosts such as Synechocystis togenerate strains that are capable of producing ethylenephototrophically. The hosts may express at least one, two, three, four,five or more copies of efe to increase ethylene production rates. Thesemethods overcome two major problems that were previously encounteredwhen using cyanobacteria to produce ethylene: poor stability of efe andpoor ethylene productivity. Without wishing to be bound by anyparticular theory, it has been discovered that removing the mutation hotspots and expressing codon-optiinized efe in hosts such as Synechocystisalleviates the metabolic burden and stability issues.

The methods disclosed herein allow for the inducible expression of efe,but also allow the utilization of stronger, constitutive promoters likepsbA to increase production rates. Other advantages include a decreasein the mutation rate within the coding region of efe and the generationof strains better able to cope with the metabolic drain imposed byethylene production. The strains generated exhibit little or noinhibition in growth while producing ethylene, nor do they exhibitstress symptoms such as yellowing that had previously been observed inother ethylene-producing strains.

The specific rate of ethylene production typically peaks 24 hours aftersubculture and then decreases. One or more components of the medium maybe a limiting factor in ethylene producing cultures. Ethylene productioncan be recovered without diluting the culture, by resuspending astationary phase culture in fresh medium. An increase in ethyleneproduction rate can also be achieved by increasing the concentration ofthe medium. For example, 10× medium may sustain high level ethyleneproduction longer than 1× medium. Peak rates of about 1500 nL mL⁻¹ hr⁻¹or greater per genomic copy of efe can be achieved after multiple roundsof resuspension. Total ethylene productivity of a culture may also beincreased on a per cell basis by growing the culture in higher lightintensities. Light intensities of about 350 μE can be reached in anincubator, but natural sunlight can be many times more intense. Higherethylene production rates may thus be reached in an outdoorphotobioreactor.

“Nucleic acid” or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides.This includes single- and double-stranded molecules (i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

Nucleic acids referred to herein as “isolated” are nucleic acids thathave been removed from their natural milieu or separated away from thenucleic acids of the genomic DNA or cellular RNA of their source oforigin (e.g., as it exists in cells or in a mixture of nucleic acidssuch as a library), and may have undergone further processing. Isolatednucleic acids include nucleic acids obtained by methods describedherein, similar methods or other suitable methods, including essentiallypure nucleic acids, nucleic acids produced by chemical synthesis, bycombinations of biological and chemical methods, and recombinant nucleicacids that are isolated.

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures that rely upon a methodof artificial replication, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes. Recombinantnucleic acids also include those that result from recombination eventsthat occur through the natural mechanisms of cells, but are selected forafter the introduction to the cells of nucleic acids designed to allowor make probable a desired recombination event. Portions of isolatednucleic acids that code for polypeptides having a certain function canbe identified and isolated by, for example, the method disclosed in U.S.Pat. No. 4,952,501.

An isolated nucleic acid molecule can be isolated from its naturalsource or produced using recombinant DNA technology (e.g., polymerasechain reaction (PCR) amplification, cloning) or chemical synthesis.Isolated nucleic acid molecules can include, for example, genes, naturalallelic variants of genes, coding regions or portions thereof, andcoding and/or regulatory regions modified by nucleotide insertions,deletions, substitutions, and/or inversions in a manner such that themodifications do not substantially interfere with the nucleic acidmolecule's ability to encode a polypeptide or to form stable hybridsunder stringent conditions with natural gene isolates. An isolatednucleic acid molecule can include degeneracies. As used herein,nucleotide degeneracy refers to the phenomenon that one amino acid canbe encoded by different nucleotide codons. Thus, the nucleic acidsequence of a nucleic acid molecule that encodes a protein orpolypeptide can vary due to degeneracies.

A nucleic acid molecule is not required to encode a protein havingprotein activity. A nucleic acid molecule can encode a truncated,mutated or inactive protein, for example. In addition, nucleic acidmolecules may also be useful as probes and primers for theidentification, isolation and/or purification of other nucleic acidmolecules, independent of a protein-encoding function.

Suitable nucleic acids include fragments or variants of SEQ ID NO:3 thatencode an ethylene-forming enzyme. For example, a fragment can comprisethe minimum nucleotides from SEQ ID NO:3 required to encode a functionalethylene-forming enzyme. Nucleic acid variants include nucleic acidswith one or more nucleotide additions, deletions, substitutions,including transitions and transversions, insertion, or modifications(e.g., via RNA or DNA analogs). Alterations may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

In certain embodiments, a nucleic acid may be identical to the sequencerepresented as SEQ ID NO:3. In other embodiments, the nucleic acids maybe least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3,or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3. Sequenceidentity calculations can be performed using computer programs,hybridization methods, or calculations. Exemplary computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, the GCG program package, BLASTN,BLASTX, TBLASTX, and FASTA. The BLAST programs are publicly availablefrom NCBI and other sources. For example, nucleotide sequence identitycan be determined by comparing a query sequences to sequences inpublicly available sequence databases (NCBI) using the BLASTN2algorithm.

Embodiments of the nucleic acids include those that encode a polypeptidethat functions as an ethylene-forming enzyme or functional equivalentsthereof. For example, the amino acid sequence of the P. syringae EFEethylene-forming enzyme is depicted in FIG. 23 and represented by SEQ IDNO:4. A functional equivalent includes fragments or variants thatexhibit the ability to function as an ethylene-forming enzyme. As aresult of the degeneracy of the genetic code, many nucleic acidsequences can encode a polypeptide having, for example, the amino acidsequence of SEQ ID NO:4. Such functionally equivalent variants arecontemplated herein.

Altered or variant nucleic acids can be produced by one of skill in theart using the sequence data illustrated herein and standard techniquesknown in the art. Variant nucleic acids may be detected and isolated byhybridization under high stringency conditions or moderate stringencyconditions, for example, which are chosen to prevent hybridization ofnucleic acids having non-complementary sequences. “Stringencyconditions” for hybridizations is a term of art that refers to theconditions of temperature and buffer concentration that permithybridization of a particular nucleic acid to another nucleic acid inwhich the first nucleic acid may be perfectly complementary to thesecond, or the first and second may share some degree of complementaritythat is less than perfect.

Nucleic acids may be derived from a variety of sources including DNA,cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Suchsequences may comprise genomic DNA, which may or may not includenaturally occurring introns. Moreover, such genomic DNA may be obtainedin association with promoter regions or poly (A) sequences. Thesequences, genomic DNA, or cDNA may be obtained in any of several ways.Genomic DNA can be extracted and purified from suitable cells by meanswell known in the art. Alternatively, mRNA can be isolated from a celland used to produce cDNA by reverse transcription or other means.

Oligonucleotides that are fragments of SEQ ID NOS:1 and 3 and antisensenucleic acids that are complementary, in whole or in part, to SEQ IDNOS:1 and 3 are contemplated herein. Oligonucleotides may be used asprimers or probes or for any other use known in the art. Antisensenucleic acids may be used, for example, to inhibit gene expression whenintroduced into a cell or for any other use known in the art.Oligonucleotides and antisense nucleic acids can be produced by standardtechniques known in the art.

Also disclosed herein are recombinant vectors, including expressionvectors, containing nucleic acids encoding ethylene-forming enzymes. A“recombinant vector” is a nucleic acid molecule that is used as a toolfor manipulating a nucleic acid sequence of choice or for introducingsuch a nucleic acid sequence into a host cell. A recombinant vector maybe suitable for use in cloning, sequencing, or otherwise manipulatingthe nucleic acid sequence of choice, such as by expressing or deliveringthe nucleic acid sequence of choice into a host cell to form arecombinant cell. Such a vector typically contains heterologous nucleicacid sequences not naturally found adjacent to a nucleic acid sequenceof choice, although the vector can also contain regulatory nucleic acidsequences (e.g., promoters, untranslated regions) that are naturallyfound adjacent to the nucleic acid sequences of choice or that areuseful for expression of the nucleic acid molecules.

A recombinant vector can be either RNA or DNA, either prokaryotic oreukaryotic, and typically is a plasmid. The vector can be maintained asan extrachromosomal element (e.g., a plasmid) or it can be integratedinto the chromosome of a recombinant host cell. The entire vector canremain in place within a host cell, or under certain conditions, theplasmid DNA can be deleted, leaving behind the nucleic acid molecule ofchoice. An integrated nucleic acid molecule can be under chromosomalpromoter control, under native or plasmid promoter control, or under acombination of several promoter controls. Single or multiple copies ofthe nucleic acid molecule can be integrated into the chromosome. Arecombinant vector can contain at least one selectable marker.

The term “expression vector” refers to a recombinant vector that iscapable of directing the expression of a nucleic acid sequence that hasbeen cloned into it after insertion into a host cell or other (e.g.,cell-free) expression system. A nucleic acid sequence is “expressed”when it is transcribed to yield an mRNA sequence. In most cases, thistranscript will be translated to yield an amino acid sequence. Thecloned gene is usually placed under the control of (i.e., operablylinked to) an expression control sequence. The phrase “operativelylinked” refers to linking a nucleic acid molecule to an expressioncontrol sequence in a manner such that the molecule can be expressedwhen introduced (i.e., transformed, transduced, transfected, conjugatedor conduced) into a host cell.

Recombinant vectors and expression vectors may contain one or moreregulatory sequences or expression control sequences. Regulatorysequences broadly encompass expression control sequences (e.g.,transcription control sequences or translation control sequences), aswell as sequences that allow for vector replication in a host cell.Transcription control sequences are sequences that control theinitiation, elongation, or termination of transcription. Suitableregulatory sequences include any sequence that can function in a hostcell or organism into which the recombinant nucleic acid molecule is tobe introduced, including those that control transcription initiation,such as promoter, enhancer, terminator, operator and repressorsequences. Additional regulatory sequences include translationregulatory sequences, origins of replication, and other regulatorysequences that are compatible with the recombinant cell. The expressionvectors may contain elements that allow for constitutive expression orinducible expression of the protein or proteins of interest. Numerousinducible and constitutive expression systems are known in the art.

Typically, an expression vector includes at least one nucleic acidmolecule encoding an ethylene-forming enzyme operatively linked to oneor more expression control sequences (e.g., transcription controlsequences or translation control sequences). In one aspect, anexpression vector may comprise a nucleic acid encoding anethylene-forming enzyme, as described herein, operably linked to atleast one regulatory sequence. It should be understood that the designof the expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of polypeptide to beexpressed.

Expression and recombinant vectors may contain a selectable marker, agene encoding a protein necessary for survival or growth of a host celltransformed with the vector. The presence of this gene allows growth ofonly those host cells that express the vector when grown in theappropriate selective media. Typical selection genes encode proteinsthat confer resistance to antibiotics or other toxic substances,complement auxotrophic deficiencies, or supply critical nutrients notavailable from a particular media. Markers may be an inducible ornon-inducible gene and will generally allow for positive selection.Non-limiting examples of selectable markers include the ampicillinresistance marker (i.e., beta-lactamase), tetracycline resistancemarker, neomycin/kanamycin resistance marker (i.e., neomycinphosphotransferase), dihydrofolate reductase, glutamine synthetase, andthe like. The choice of the proper selectable marker will depend on thehost cell, and appropriate markers for different hosts as understood bythose of skill in the art.

Suitable expression vectors may include (or may be derived from) plasmidvectors that are well known in the art, such as those commonly availablefrom commercial sources. The Examples below illustrate the constructionof exemplary expression vectors containing an ethylene-forming enzyme.Vectors can contain one or more replication and inheritance systems forcloning or expression, one or more markers for selection in the host,and one or more expression cassettes. The inserted coding sequences canbe synthesized by standard methods, isolated from natural sources, orprepared as hybrids. Ligation of the coding sequences to transcriptionalregulatory elements or to other amino acid encoding sequences can becarried out using established methods. A large number of vectors,including bacterial, yeast, and mammalian vectors, have been describedfor replication and/or expression in various host cells or cell-freesystems, and may be used with the secretion sequences described hereinfor simple cloning or protein expression.

Certain embodiments may employ cyanobacterial promoters or regulatoryoperons. For example, a promoter may comprise an rbcLS operon ofSynechococcus, a cpc operon of Synechocystis sp. strain PCC 6714, thetRNApro gene from Synechococcus, the nirA promoter from Synechococcussp. strain PCC 7942, which is repressed by ammonium and induced bynitrite. The efficiency of expression may be enhanced by the inclusionof enhancers that are appropriate for the particular cyanobacterial cellsystem which is used, such as those described in the literature.Suitable promoters also include the petE and psbA promoters.

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transformednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, integration of the nucleic acid molecules intoone or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals (e.g., ribosomebinding sites), modification of nucleic acid molecules to correspond tothe codon usage of the host cell, and deletion of sequences thatdestabilize transcripts.

The nucleic acids, including parts or all of expression vectors, may beisolated directly from cells, or, alternatively, the polymerase chainreaction (PCR) method can be used to produce the nucleic acids. Primersused for PCR can be synthesized using the sequence information providedherein and can further be designed to introduce appropriate newrestriction sites, if desirable, to facilitate incorporation into agiven vector for recombinant expression. The nucleic acids can beproduced in large quantities by replication in a suitable host cell(e.g., prokaryotic or eukaryotic cells such as bacteria, yeast, insector mammalian cells). The production and purification of nucleic acidsare described, for example, in Sambrook et al., 1989; F. M. Ausubel etal., 1992, Current Protocols in Molecular Biology, J. Wiley and Sons,New York, N.Y.

The nucleic acids described herein may be used in methods for productionof AKG, pyruvate or ethylene through incorporation into cells, tissues,or organisms. In some embodiments, a nucleic acid may be incorporatedinto a vector for expression in suitable host cells. Alternatively,gene-targeting or gene-deletion vectors may also be used to disrupt orablate a gene. The vector may then be introduced into one or more hostcells by any method known in the art. One method to produce an encodedprotein includes transforming a host cell with one or more recombinantnucleic acids (such as expression vectors) to form a recombinant cell.The term “transformation” is generally used herein to refer to anymethod by which an exogenous nucleic acid molecule (i.e., a recombinantnucleic acid molecule) can be inserted into a cell, but can be usedinterchangeably with the term “transfection.”

Non-limiting examples of suitable host cells include photosyntheticbacteria, green algae, and cyanobacteria, including naturallyphotosynthetic microorganisms or engineered photosyntheticmicroorganisms. Exemplary microorganisms that are either naturallyphotosynthetic or can be engineered to be photosynthetic include, butare not limited to, bacteria; fungi; archaea; protists; eukaryotes, suchas a green algae; and animals such as plankton, planarian, and amoeba.Examples of naturally occurring photosynthetic microorganisms includeSpirulina maximum, Spirulina platensis, Dunaliella salina, Botrycoccusbraunii, Chlorella vulgaris, Chlorella pyrenoidosa, Serenastrumcapricomutum, Scenedesmus auadricauda, Porphyridium cruentum,Scenedesmus acutus, Dunaliella Scenedesmus obliquus, Anabaenopsis,Aulosira, Cylindrospermum, Synechoccus sp., Synechocystis sp., and/orTolypothrix.

In exemplary embodiments, the host cell may be a microbial cell, such asa cyanobacterial cell, and may be from any genera or species ofcyanobacteria that is genetically manipulable. Examples of suitablecyanobacteria include the genus Synechocystis (e.g., strains such asSynechocystis sp. PCC 6803), Synechococcus, Thermosynechococcus, Nostoc,Prochlorococcu, Microcystis, Anabaena, Spirulina, and Gloeobacter.

Further examples of cyanobacteria suitable for use as host cells in themethods described herein include Chroococcales cyanobacteria from thegenera Aphanocapsa, Aphanothece, Chamaesiphon, Chroococcus,Chroogloeocystis, Coelosphaerium, Crocosphaera, Cyanobacterium,Cyanobium, Cyanodictyon, Cyanosarcina, Cyanothece, Dactylococcopsis,Gloecapsa, Gloeothece, Merismopedia, Microcystis, Radiocystis,Rhabdoderma, Snowella, Synychococcus, Synechocystis,Thermosenechococcus, and Woronichinia; Nostacales cyanobacteria from thegenera Anabaena, Anabaenopsis, Aphanizomenon, Aulosira, Calothrix,Coleodesmium, Cyanospira, Cylindrospermosis, Cylindrospermum, Fremyella,Gleotrichia, Microchaete, Nodularia, Nostoc, Rexia, Richelia, Scytonema,Sprirestis, and Toypothrix; Oscillatoriales cyanobacteria from thegenera Arthrospira, Geitlerinema, Halomicronema, Halospirulina,Katagnymene, Leptolyngbya, Lintnothrix, Lyngbya, Microcoleus,Oscillatoria, Phormidium, Planktothricoides, Planktothrix, Plectonema,Pseudoanabaena/Limnothrix, Schizothrix, Spirulina, Symploca,Trichodesmium, and Tychonema; Pleurocapsales cyanobacteria from thegenera Chroococcidiopsis, Dermocarpa, Dermocarpella, Myxosarcina,Pleurocapsa, Stanieria, and Xenococcus; Prochlorophytes cyanobacteriafrom the genera Prochloron, Prochlorococcus, and Prochlorothrix; andStigonentatales cyanobacteria from the genera Capsosira, Chlorogeoepsis,Fischerella, Hapalosiphon, Mastigocladopsis, Nostochopsis, Stigonema,Symphyonema, Symphonemopsis, Umezakia, and Westiellopsis. In certainembodiments, the host cell may be from the genus Synechococcus, such asSynechococcus bigranulatus, Synechococcus elongatus, Synechococcusleopoliensis, Synechococcus lividus, Synechococcus nidulans, andSynechococcus rubescens.

Host cells can be transformed, transfected, or infected as appropriatewith gene-disrupting constructs or plasmids (e.g., an expressionplasmid) by any suitable method including electroporation, calciumchloride-, lithium chloride-, lithium acetate/polyethylene glycol-,calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake,spheroplasting, injection, microinjection, microprojectile bombardment,phage infection, viral infection, or other established methods.Alternatively, vectors containing a nucleic acid of interest can betranscribed in vitro, and the resulting RNA introduced into the hostcell by well-known methods, for example, by injection. Exemplaryembodiments include a host cell or population of cells expressing one ormore nucleic acid molecules or expression vectors described herein (forexample, a genetically modified microorganism). The cells into whichnucleic acids have been introduced as described above also include theprogeny of such cells.

Vectors may be introduced into host cells such as cyanobacteria bydirect transformation, in which DNA is mixed with the cells and taken upwithout any additional manipulation, by conjugation, electroporation, orother means known in the art. Expression vectors may be expressed bycyanobacteria or other host cells episomally or the gene of interest maybe inserted into the chromosome of the host cell to produce cells thatstably express the gene with or without the need for selective pressure.For example, expression cassettes may be targeted to neutralcyanobacterial chromosomal sites by double recombination. In certainembodiments, the gene encoding the ethylene-forming enzyme is stable inthe host cell for greater than 4, greater than 10, greater than 25,greater than 50 or greater than 100 passages. In some embodiments, thehost cell expresses a function copy of the ethylene-forming enzyme forgreater than 4, greater than 10, greater than 25, greater than 50 orgreater than 100 passages.

Host cells with targeted gene disruptions or carrying an expressionvector (i.e., transformants or dories) may be selected using markersdepending on the mode of the vector construction. The marker may be onthe same or a different DNA molecule. In prokaryotic hosts, thetransformant may be selected, for example, by resistance to ampicillin,tetracycline or other antibiotics. Production of a particular productbased on temperature sensitivity may also serve as an appropriatemarker.

Host cells may be cultured in an appropriate fermentation medium. Anappropriate, or effective, fermentation medium refers to any medium inwhich a host cell, including a genetically modified microorganism, whencultured, is capable of producing AKG, pyruvate or ethylene. Such amedium is typically an aqueous medium comprising assimilable carbon,nitrogen and phosphate sources, but can also include appropriate salts,minerals, metals and other nutrients. Microorganisms and other cells canbe cultured in conventional fermentation bioreactors or photobioreactorsand by any fermentation process, including batch, fed-batch, cellrecycle, and continuous fermentation. The pH of the fermentation mediumis regulated to a pH suitable for growth of the particular organism.Culture media and conditions for various host cells are known in theart. A wide range of media for culturing cyanobacteria, for example, areavailable from ATCC.

Photosynthetic microorganisms may be cultured according to techniquesknown in the art. For example, cyanobacteria may be cultured orcultivated according to techniques known in the art such asphotobioreactor based techniques. One example of typical laboratoryculture conditions for cyanobacterium is growth in BG11 medium (ATCCMedium 616) at 30° C., and 5% CO₂ under 60 μEm⁻²s⁻¹ constantillumination from fluorescent bulbs, with shaking for liquid cultures orwithout shaking for plates.

Methods for producing AKG, pyruvate or ethylene are also disclosedherein. Cells may be cultured as described above and exposed to a carbonsource and light for production of AKG, pyruvate or ethylene. Carbonsources include atmospheric carbon dioxide or carbon dioxide providedfrom an artificial source such as a compressed storage tank. Culturesmay also be provided with additional carbon sources such as sugars(e.g., glucose and other saccharides). For phototrophic AKG, pyruvate orethylene production, cells may be exposed to either artificial light ornatural light such as sunlight. Nitrogen starvation may be achieved byany conventional technique, such as resuspending cells in mediacontaining little or no nitrogenous compounds, or by replenishingdepleted media with fresh media containing little or no nitrogenouscompounds.

As used herein, “nitrogen starvation conditions” refers to culturingcells in media that is substantially free of nitrogen-containingcompounds, for example, culturing cells in nitrogen-depleted media.Nitrogen depleted media or conditions that are substantially nitrogenfree refers to media or conditions wherein nitrogen ions are present atlevels less than at most 200 μM. Examples include nitrogen levels of200, 150, 100, 50, 25 or 10 μM or less. In certain embodiments, cellsgrown in nitrogen-containing media may be subsequently cultured innitrogen-depleted media (for example, by isolating the cells andresuspending the cells in nitrogen-depleted media, with or withoutwashing the cells in nitrogen-depleted media prior to the resuspension.In some embodiments, cells cultured in nitrogen-containing media may besupplemented with fresh nitrogen-depleted media as the cultureprogresses.

Nitrogen-containing compounds may be added to cells growing innitrogen-depleted media to facilitate cell growth. In certainembodiments, nitrogen-containing compounds may be added to cell culturesto a final concentration of less than about 1 mM, or less than about900, 800, 700, 600, 500, 400, 300, 200, or 100 μM.

Cell densities, light intensities, medium concentrations, mediumcompositions and other culture conditions may be varied to achieve peakAKG, pyruvate or ethylene production rates and/or sustain AKG, pyruvateor ethylene production at peak rates. Exemplary light conditionssuitable for cell cultures include growth under light (e.g., whitelight) of at least about 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, or 700 μE m⁻² s⁻¹. Even higher light (e.g., up toabout 2000 μE m⁻² s⁻¹) may be suitable for dense cultures, typicallywith mixing. Examples include 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, or 2000 μE m⁻² s⁻¹

In certain embodiments, AKG or pyruvate culture concentrations may be atleast 100 mg L⁻¹. Additional culture concentrations of AKG or pyruvatethat may be attained using the methods herein include at least 10, 20,50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg L⁻¹. In someembodiments, AKG concentrations may be greater than 1000 mg L⁻¹. Forexample, AKG concentrations may be at least about 1000, 1500, 2000,2500, 3000 or 3500 mg L⁻¹. Pyruvate concentrations may be at least about1, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g L⁻¹. The peak ethyleneproduction rate is the highest rate of ethylene production attained fora given culture over a given time period. In certain embodiments,ethylene is produced at a peak production rate of at least 500 mL mL⁻¹hr⁻¹. Additional peak production rates that may be attained using themethods herein include ethylene is produced at a peak production rate ofat least about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, or 300 μL mL⁻¹hr⁻¹. In some embodiments, ethylene may be produced at a peak rategreater than 200 μL mL⁻¹ hr⁻¹.

Cells may be cultured in batch fermentations and cells that reachstationary phase may be resuspended in fresh medium to continue ethyleneproduction. The medium may be 1× in concentration or may be increasedanywhere from 1× to 5× to 10× or greater to support increasedproduction. Alternatively, medium components may be added back to thecultures as they are depleted by the culture and/or waste products maybe removed from the cultures as they are produced.

AKG or pyruvate produced by the cell cultures may be released from thecells and collect in the culture media. AKG or pyruvate may be harvestedor isolated from the cultures using any means known in the art. Forexample, the cells may be separated from the culture media and AKG orpyruvate separated from the culture supernatant via conventionalseparation techniques. Cells may also be lysed and AKG or pyruvateisolated from the cell lysates via conventional separation techniques.

Ethylene produced by the cell cultures is released from the cells andcollects in the head space of the culture vessel. Ethylene may beharvested from the cultures using any means known in the art. Forexample, the culture vessel may contain collection piping to removeethylene from the head space by active or passive processes. Culturevessel piping may be employed to both provide carbon dioxide to thecells and to remove ethylene produced by the cultures.

EXAMPLES Example 1

The following materials and methods were used in subsequent Examplesdetailed below.

Culturing Conditions

Synechocystis was cultured at 30° C. in BG11 media (phycology medium,Sigma-Aldrich, USA) supplemented with 20 mM TES buffer and 100 mM NaHCO₃(with or without 3 μM NiCl₂) under a constant light flux ofapproximately 50 to 60 μE m⁻² s⁻¹ supplied by cool white fluorescentlamps. Cultures were shaken (liquid cultures; plates were not shaken)and either bubbled with 2% CO₂ in air or under a 5% CO₂ headspace inair. When appropriate, plates were supplemented with 50 μg/mLspectinomycin and liquid cultures were supplemented with 25 μg/mLspectinomycin. The antibiotics gentamicin and kanamycin were used at 5and 50 μg/mL in liquid cultures for inoculation from plates. Thereafter,the antibiotics were removed for growth of cultures in BG11. The samegrowth conditions were used for nitrogen starved cultures, except thatthe cells were resuspended in BG11-N media, which is identical to theBG11 medium except that the NaNO₃ is replaced with NaCl (mole:mole).Cells were harvested from log-phase cultures in replete medium (OD₇₃₀,0.6-0.8; DW, 0.3-0.5 g/L) by centrifugation, washed, resuspended inBG11-N and placed under the same light and atmosphere conditions asabove.

Optical density was measured at 730 nm with a Biowave II UV/Visspectrophotometer (Biochrom, Inc., Cambridge, England). Absorbance ofwhole-cell suspensions of cultures were monitored in a wavelength rangeof 300-800 nm each day with a spectrophotometer (DU800, Beckman Coulter,USA). Concurrent measurements of dry weight were measured by passing 5mL culture through 0.45 micron pre-weight filters (Pall Corporation,USA), which were subsequently dried to constant mass at 55° C. forseveral days and re-weighed.

E. coli was grown using standard procedures in LB medium at 37° C.,supplemented with 50 μg/mL spectinomycin as appropriate.

Glycogen Content

Glycogen content was measured as described by Ernst et al. (Archives ofMicrobiology 140:120-125 (1984)). Briefly, 1-2 mL of cells were pelletedby centrifugation at 13,000×g for 5 minutes. To each pellet, 200 μL ofaqueous KOH (40% by weight) was added. The alkaline suspensions werevortexed and then incubated at 100° C. for 1 hour. Subsequently, 600 μLof cold (0° C.) absolute ethanol was added and this suspension and wascentrifuged at 13,000×g. The supernatant was discarded and the pelletwas washed with cold ethanol, dried under air at 70° C., and suspendedin 1 mL of 200 mM sodium acetate buffer (pH 4.75) containing 10 unitsamyloglucosidase (Sigma). This solution was incubated at 37° C. for atleast 5 hours after which glucose concentrations were determined by aglucose assay kit, which utilizes hexose kinase and NADH (Sigma).Glycogen recovery by this method with known quantities of bovineglycogen (Sigma) was greater than 95%.

Dry Weight Determination

Clear glass bottles (Wheaton, USA) with an approximate capacity of 25 mLwere cleaned, acid/base washed, and placed at 55° C. for drying forseveral weeks. Exactly 50 mL of cells were harvested each day,transferred to 50 mL conical tubes, and centrifuged for 10 minutes at4,000×g in a swinging bucket rotor. Supernatants were carefullydecanted, and approximately 5 mL of deionized water was used toresuspend each of the remaining cell pellets and rinse residual materialfrom conical tubes. Cell suspensions and rinses were transferred to apre-weighed glass bottle, which was returned to a 55° C. oven forseveral weeks until all moisture had evaporated (indicated by constantcool dry mass). Weights were measured once for each culture per day.Error bars represent 3 separate cultures.

Photosynthetic Fluorescence Parameters and O₂ Evolution

Light-adapted variable fluorescence ΔFv/Fm′ measures the effectivequantum efficiency of cyanobacteria assuming that the phycobilin contentof the cells is similar. A Closed Fluorocam FC-800 C (Photon SystemInstruments, Czech Republic) was used to measure ΔFv/Fm′ daily withrespect to nitrogen starvation time under approximately 100 μE m⁻² s⁻¹light for wild-type and AGP-cultures.

Oxygen Evolution

Whole-chain oxygen evolution for wild-type and AGP-cultures weremeasured electrochemically with an oxygen electrode system (PhotonSystem Instruments) using a custom microelectrode from Microelectrodes,Inc (New Hampshire, USA). Samples were prepared by centrifuging 7 mLcells from culture and resuspending in 3.5 mL photosynthesis bufferimmediately before each time point was collected. Photosynthesis bufferconsisted of 20 mM TES, 100 mM sodium bicarbonate, and 1 mM potassiumphosphate. Oxygen concentrations were determined every 100 ins, and theslope of the rise in oxygen concentration in the presence of saturatingred light was taken as the relative whole-chain oxygen evolution rate,which was normalized by the difference in voltage between air-saturatedbuffer and dithionite-treated (anaerobic) buffer.

Intracellular Metabolite Concentrations

To determine metabolite concentrations, 2 mL of cells were centrifugedat 13,000×g for 2 minutes. The cell pellet was then placed on dry iceafter decanting the supernatant. Pellets were stored at −80° C. untilextraction. For extraction, 2 mL of boiling 70% ethanol in water waspoured over the frozen pellet. The mixture was then incubated at 100° C.for 4 minutes and subsequently centrifuged at 13,000×g for 4 minutes.The supernatant in 70% ethanol was dried at 37° C. under a steady streamof nitrogen gas, followed by adding 40 μL of 20 mg/mL methoxyaminehydrochloride in pyridine. The resulting suspension was vortexed for 2minutes and subsequently incubated at 37° C. for 90 minutes, sealed withTeflon-coated rubber screw-caps. Then, 60 μL MSTFA(N-methyl-N-trimethylsilyltrifluoroacetamide)+1% TMCS(Trimethylchlorosilane) was added to the suspension, followed byvortexing, and sealed incubation for 30 minutes at 55° C.

One microliter of the resulting suspension was injected into a GC/MS(gas chromatograph/mass spectrometer) using a splitless injector. Thesystem used a 7890A GC system and a 5975C inert XL MSD (mass selectivedetector) with a Tripple Axis Detector (Agilent Technologies Inc., USA).Inlet temperature was 225° C. and compounds were separated using a 30 inDB-35MS column (Agilent Technologies Inc.) with Helium carrier gas and aflow rate of 1 mL/min. GC parameters included 50° C. isothermal heatingfor 2 minutes followed by a 5° C./min increase to 150° C., a hold for 2minutes at 150° C., and a second temperature ramping phase of 7° C./minto 320° C. and a 2 minute hold at 320° C. The MSD transfer line wasmaintained at 280° C. The MS quadrupole and MS source temperature weremaintained at 150° C. and 230° C., respectively. Compounds were detectedusing the scan mode with a mass detection range of 40-500 atomic massunits (amu).

Chromatograms were analyzed with MSD Enhanced ChemStation data analysissothvare (Agilent Technologies Inc.). Identities of compounds were basedon retention time and MS spectral parameters from pre-run standardcompounds. Unique m/z ions (target ions) were selected for each compoundfor manual quantification based on target ion peak areas. Concentrationamounts were estimated based on the target ion peak area of the signalrelative to this peak response of the pre-run standard at aconcentration of 0.1 mg/mL.

Measuring Extracellular AKG Concentrations

High-performance liquid chromatography (HPLC) was used to measure alphaketoglutarate (AKG) concentrations. Briefly, 2 mL cell suspensions fromcultures were taken and passed through a 0.45 μM filter (PallCorporation, USA). The resulting cell-free solution was used forinjection (100 μL) into an Agilent 1200 HPLC. The HPLC was run at 45° C.with a 4 mM aqueous sulfuric acid mobile phase and a Biorad HPLC OrganicAcid Analysis (Aminex HPX-87H Ion exchange) column with a refractiveindex detector. AKG gave a linear response between a concentration rangeof 25 μM and 10 mM.

The identity of AKG was also confirmed by GC/MS and proton nuclearmagnetic resonance (NMR) spectroscopy. The GC/MS protocol forextracellular media was used as described above, except cell media (2mL) was dried under nitrogen, rather than a 70% cell extract. Proton NMRwas measured by drying 2 mL of cell extract in 70% ethanol followed byaddition of 1 mL deutorated water (Sigma). From the resulting solution,600 μL was added to a pyrex NMR tube (5 mm diameter, 7 inch length). Thesample was measured with 512 scans in a 500 MHz Varian Spectrometer.

Carbon-13 Isotope Labeling

Cells cultured in (nutrient replete) BG11 were resuspended in BG11-Nmedium with some sodium bicarbonate replaced with ¹³C sodium bicarbonate(Sigma-Aldrich, USA) as indicated by percentages (final concentration of¹²C+¹³C sodium bicarbonate, 100 mM). Cell-free medium following 1-day ofgrowth in BG11-N was collected, and proton NMR spectra were determinedas above. Carbon spectra were collected on a 400-MHz Varian spectrometerat 25° C. (18,000 scans with a relaxation delay of 1 s). All spectrawere processed using MestReNova software v 6.0.4 (Mestrelab ResearchS.L., Santiago de Compostela, Spain).

Ethylene Production Assay

Stationary phase cultures were diluted to a starting OD₇₃₀ ofapproximately 0.2. Each day for the duration of the experiment 2 mL ofculture was transferred into a 13 mL Hungate tube and incubated for 4hours while shaking in 60 μm⁻²s⁻¹ light. The ethylene produced was thenquantified using gas chromatography.

Example 2 Mutagenesis and Creation of Mutant Strains

As depicted in FIG. 12, fusion PCR was used to create a gene deletionDNA fragment that was composed of approximately 600 by immediatelyupstream the glgC gene fused to a DNA fragment of the genR codingsequences from the pUC119 DNA followed by approximately 600 byimmediately downstream the glgC gene. This construct was transformedinto a glucose tolerant strain of Synechocystis and selected over timeon standard BG11 agar plates containing gentamicin. Transgenic lineswere screened by PCR followed by western blot and glycogen assay for acomplete depletion of the glgC gene, AGP protein and glycogen,respectively. Herein we refer to the generated mutant strain as theAGP-strain as an indication that this strain lacks an ADP-glucosepyrophosphorylase.

A specific ORF replacement of the glgC gene (ski 176) with agentamicin-resistance gene was performed using homologous recombination.A gene deletion construct was created using fusion PCR that containedflanking regions of the glgC gene and the pUC119-gen plasmid (PCRprimers described in Table 1), using KOD Hot Start DNA Polymerase(Novogen) under standard conditions. Transformation of wild type wasperformed using the created gene-deletion construct. Selection wasperformed on agar plates with BG-11 (10 mM NaHCO₃, gentamicin between 20and 50 μg/mL). Segregation of the mutation was verified by PCR productanalysis (FIG. 13) using primers listed in Table 1. Glycogendetermination, glucosyiglycerol determination by HPLC, and western blotprotein analysis demonstrated that the glgC mutant is a fully segregatedmutant. For western blotting, protein was isolated from logarithmicallygrowing cultures, extracted, and quantified. Proteins were separatedusing Mini Protean TGX Gels (Biorad), and blotted using Fast Semi DryBlotter (Pierce) onto PVDF (Biorad) membranes. A custom peptide primaryantibody for GlgC (YenZym Antibodies, LLC) was used in conjunction withGoat Anti-Rabbit secondary antibody (Pierce) and a CN/DAB Substrate Kit(Thermo Scientific).

Synechocystis sp. PCC 6803 strain ΔglgC psbA2::glgC was constructed fromthe ΔglgC line above with the pPSBA2KS vector altered by removal of theSalI site via partial digest and blunting to allow for retention of thekanamycin resistance gene. The glgC gene was amplified from genomic DNAisolated from WT by PCR and inserted into the vector between the NdeIand SalI restriction sites. Transformation was conducted by incubationof approximately 1 μg of the integration vector for 6 hours with 200 μLcells (adjusted to optical density of 2.5 from logarithmic-phasecultures), followed by addition of 2 mL BG11, 24 hours outgrowth inculture tubes under standard growth conditions, and plating of 200 μL onBG11 plates with 200 μg/mL kanamycin. The mutation was verified by PCRproduct analysis using primers listed in Table 1.

TABLE 1 SEQ ID NOs Fusion PCR Primers 5′agpF GTCATGCCAATGCCGTTATCSEQ ID NO: 5 agp/gn atgR CATCGTTGCTGCTGCGTAACATTTCGAAGTCAAGTTTAGAACAGAGGSEQ ID NO: 6 agp/gn atgF CCTCGGTTCTAAACTTGACTTCGAAATGTTACGCAGCAGCAACGATG SEQ ID NO: 7agp/gn taaR  GTGCGAGGAAAGAAACTGGCCTAAGGTGGCGGTACTTGGGTCG SEQ ID NO: 8agp/gn taaF CGACCCAAGTACCGCCACCTAAGGCCAGTTTCTTTCCTCGCAC SEQ ID NO: 93′agpR GGTGAACGACAAAGCCAGTTA SEQ ID NO: 10 Genomic Segregation Screening5′outagpF CAGATGGCCCGCTGTTTATT SEQ ID NO: 11 agpR AACAACCAGAGGTATTGCCGSEQ ID NO: 12 GentintR AAGAAGCGGTTGTTGGCGC SEQ ID NO: 13 PsbA2outFCCCATTGCCCCAAAATACATC SEQ ID NO: 14

Example 3

Growth properties of Cyanobacterial Strains

As shown in FIG. 2A, the growth rates of wild-type and AGP-cultures infiill-nitrate medium are nearly identical. However, these strains behavedifferently under nitrogen starvation (FIGS. 2B and C). Wild-typecultures of Synechocystis 6803 nearly double in optical density (FIG.2B) before reaching a steady state over 5 days of incubation in BG11-N.This behavior mimics the trends seen for dry weight measurements takenover a period of nitrogen arrest (FIG. 9). The AGP-strain remainsblue-green over this period of time, and does not increase in opticaldensity or cell dry weight. Nitrogen starvation of Synechocystis sp. PCC6803 and other non-diazotrophic cyanobacteria typically leads to areduction in phycobilin proteins, resulting in a culture color changefrom blue-green to yellow-green, a process sometimes referred to as“bleaching”.

Example 4 Cyanobacterial Strain Characteristics

FIG. 3 displays glycogen content as a percentage of cell dry weight inboth wild-type (WT) and AGP-strains under growth in BG11 or incubationin BG11-N. No detectable amounts of glycogen were observed in theAGP-strain under either condition (detection limit 0.2% of dry weight),but the glycogen content of wild-type cells nearly tripled after 3 daysof incubation in BG11-N. That the optical densities and dry weights(FIGS. 8 and 9) of AGP-cultures do not substantially change over thecourse of nitrogen starvation suggests two possibilities: (1)photosynthesis of the AGP-strain fixes no (or very little) net carbon,or (2) the AGP-strain is producing another product from its fixed carbonthat does not contribute to cell dry weight (e.g. is excreted into themedium).

To investigate these two possibilities, the effective quantum efficiencyof light-adapted cells (a relative indicator of photochemical reactions)was examined under a constant light source (FIG. 4). This measurementcan be reliably obtained from cyanobacterial cells that do not changephycobilin content, which appeared to be the case for the AGP-strain, asthe color of these cultures does not change with respect to time, andthe absorbencies of whole-cell suspensions at 630 nm (phycobilins) and680 nm (chlorophyll) do not change substantially in the AGP-strain (FIG.10). The light-adapted quantum efficiency (ΔF/Fm′) for AGP-cells remainsrelatively constant for up to 5 days incubation in BG11-N (FIG. 4). Thismay indicate that photosynthesis under non-saturating light (˜100 μE m⁻²s⁻¹) leads to continuous fixation of CO₂ at the same rate atnon-nitrogen starved conditions (time=0 days) as after 5 days ofnitrogen starvation (time=5 days).

The relationship between photosynthetic capacity and nitrogen starvationwas also examined. Whole-chain oxygen evolution under saturating lightconditions was measured for wild-type and AGP-cultures (FIG. 5).Evolution rates are reported as a percentage of non-nitrogen starvedcultures (day 0 cultures), but evolution rates normalized to chlorophyllcontent at this time point were within error (˜200 μmol O₂ mgchla⁻¹hr⁻¹). Normalizing to Chlorophyll content was not appropriate forcultures starved of nitrogen for any period of time, as chlorophyllconcentrations degrade slightly with nitrogen starvation, which wouldartificially increase apparent evolution rates. FIG. 5 shows that O₂evolution rates of both wild-type and AGP-cultures decline. However, theAGP-cultures decline at a rate similar to or slower than that of thewild-type. These results indicate that AGP deletion also causes anon-bleaching phenotype under nitrogen starvation, but does not lead tosignificantly higher decline in photosynthesis capacity relative to thewild-type strain. This in turn suggests that the cells are fixing CO₂and directing the resulting photosynthate into a product that does notcontribute to dry weight.

Example 5 AKG Production

Intracellular concentrations of some metabolites in both wild-type andAGP-strains were measured by GC/MS for log-phase (nitrogen replete) andnitrogen starved (2-days) cultures. Previous results suggested that afraction of photosynthate may have been redirected into sucrose, a knownsecondary osmolyte, which has been shown to increase in somesalt-treated cells of Synechocystis 6803. FIG. 6A shows that this is notthe case, as the AGP-cultures in both N+ and N− conditions have lesssucrose than wild-type strains. Measurements of some citric acid cycleintermediates (citric acid, fumaric acid, and alpha ketoglutarate)indicate that flux has instead been directed (at least in part) to thecitric acid cycle in APG-cultures lacking nitrogen (FIGS. 6B-D). In theabsence of available nitrogen from growth media (in the form ofammonium, nitrate, or urea) or from phycobilin degradation, AKG, whichis produced irreversibly from isocitrate via isocitrate dehydrogenase,is a “dead end” product. In other words, AKG apparently cannot befurther metabolized by cells in the absence of NH₄ ⁺, and thereforeaccumulates to high concentrations. In fact, AKG is known to be thesignaling molecule for sensing carbon:nitrogen ratio statusintracellularly in cyanobacteria.

Examination of extracellular media by HPLC revealed a substantialaccumulation of AKG in nitrogen-starved AGP-cultures (FIG. 7). Theidentity of this extracellular metabolite was confirmed by GC/MS (ofderivatized dried cell media). Proton NMR spectra of ethanol extracts ofnitrogen-starved AGP-cells also showed a high abundance of AKG. Forapproximately 4-5 days of incubation in BG11-N, linear amounts of AKGare produced—consistent with the observation that low-lightphotosynthesis effective quantum yield is constant for this period oftime (FIG. 4).

FIG. 8 illustrates the redistribution of mass from cellular constituentsin the wild-type strain, to excreted alpha ketoglutarate in theAGP-strain. While the total amount of material made photosyntheticallyfrom the AGP-strain (sum of stacked values) is similar to that ofwild-type dry weight, approximately 50% of the total amount of mass isin the form of one excreted product in the AGP-strain: alphaketoglutarate.

Example 6 Pyruvate Production

Proton NMR studies indicating the presence of AKG also revealed twoadditional peaks in this material, at ppm shifts of 2.38 and 1.44relative to the NMR internal standard trimethylsilyl propionate, TSP. Wedesigned a medium that is proton NMR silent. That is, the cells weresuspended in a medium that does not have abundant peaks by proton NMRand incubated under standard nitrogen starvation conditions. (Ourstandard BG-11 medium uses TES buffer, citric acid, and ETDA, all ofwhich have proton NMR signatures; the new buffer is standard BG-11 withNaNO₃ replaced with NaCl, mole for mole, and with TES, citric acid, andNa₂EDTA removed with no substitutions). In doing so, additional peakswere observed in this buffer that grew with respect to days of nitrogenstarvation.

The predominant peak is at a shift (with respect to TSP) of 2.36 ppm.This peak can be matched to either succinate or pyruvate in proton NMRlibraries. However, an additional peak is found at 1.47 ppm that is notpresent in succinate standards. Samples of NMR-silent buffers in whichcells had been nitrogen starved revealed a new peak, previouslyunidentified because it has the same retention time as TES. A standardof sodium pyruvate was prepared in this buffer and had a matchingretention time. Thus, HPLC and proton NMR analyses confirmed thatpyruvate is a second metabolite produced by AGP-cells under nitrogenstarvation. Pyruvate and AKG account for approximately 85+/−11% of fixedcarbon.

Example 7 Construction of Stable, Ethylene Producing Strains ofSynechocystis

To generate petE:EFE, a modified P syringae efe gene was synthesized inpUC57 vector. The efe coding region was excised from pUC57 with LguI andtransferred to pPETE cut with the same enzyme. This placed efe under thecontrol of the plasmid-born petE promoter. This plasmid was namedpJU101. NU101 was then integrated into the chromosome of Synechocystisvia double recombination into the slr0168 region as previously described(Zang et al., J. Microbiol, 45:241-245 (2007)). To generate psbA:EFE,the 5′ end of the modified efe was synthesized, with the first 75 by ofthe pea plant chloroplast psbA promoter attached in vector pUC57. ThepsbA promoter and efe coding region were excised from pUC57 with Sinaiand BsrGI and transferred to NU101 cut with the same enzymes. Thisrestored the full coding region of efe while simultaneously replacingthe petE promoter with the psbA promoter. This plasmid, named pJU102,was then integrated into the chromosomes of Synechocystis via doublerecombination into the slr0168 region.

To make a second plasmid for integration into an alternate genomic site,efe was cloned from pJU102, cut with EcoRI and XhoI, and placed into theSalI and NcoI sites of pPSBA2KS. This plasmid, named pJU112, was thenintegrated into the chromosomes of the Synechocystis containing thefirst copy of efe via double recombination into the psbA2 locus togenerate the 2×psbA:Sy-efe strain.

Previous attempts to engineer cyanobacteria to produce ethylene wereunsuccessful because the efe gene was readily inactivated within threegenerations of successive culture growth (see Takahama et al., J.Biosci. Bioeng. 95:302-305 (2003)). Inactivation of the efe geneappeared to result from specific duplications at certain mutation hotspots within the gene, leading to truncated peptides. To overcome thisstability issue, several silent mutations were made in the P. syringaeefe sequence that eliminated the potential mutation hotspots whileretaining the correct amino acid sequence of EFE. In addition, wecodon-optimized the sequence of efe for improved expression inSynechocystis.

The nucleic acid sequence of the modified efe gene is shown in FIG. 22and is represented as SEQ ID NO:3. The amino acid sequence of theproduct of the modified efe gene is shown in FIG. 23 and is representedas SEQ ID NO:4. The modified efe was placed under the control of eitherthe copper regulated petE promoter or the constitutive pea plant psbApromoter. The efe gene and an accompanying spectinomycin resistancecassette were inserted into the chromosome of Synechocystis at a neutralsite (s/r0168) via double recombination.

A second source of instability of efe in previous studies was theapparent metabolic burden that ethylene production imposed on theorganism. Previous work showed that EFE significantly reduced a strain'sspecific growth rate, thereby applying a strong selection pressure forcells that harbor non-functional copies of efe (see Sakai et al., J.Ferment. Bioeng. 84:434-443 (1997) and Takahama et al., J. Biosci.Bioeng. 95:302-305 (2003)). Colonies expressing a functional copy of efealso appeared yellow, indicating stress caused be expression of the efegene. The modified efe gene was expressed in Synechocystis and norepression of growth rate was observed (see FIG. 15), suggesting no orless metabolic burden resulting from EFE. This reduced selection againstfunctional copies of efe, resulting in increased stability of efe in theefe-expressing Synechocystis strains. Additionally, ethylene-producingcolonies of Synechocystis appeared indiscernible from wild-type coloniesof the same strain, further suggesting decreased metabolic stress fromEFE expression.

Example 8

Ethylene Production Driven by the petE and psbA Promoters

The modified efe was initially expressed under the control of thecopper-inducible petE promoter to impart stability to the system by notinducing expression of efe until stationary phase. For comparison,modified efe was also expressed from the pea plant chloroplast genomepsbA constitutive promoter. Ethylene production rates of the tworecombinant strains were compared using gas chromatography and ethyleneproduction was observed to peak early in growth and then fall off as theculture enters stationary phase (see FIG. 16A). Diluting the stationarycultures with fresh medium reinitiated log phase growth and restoredpeak ethylene production rates (See FIG. 16B). These data suggest thatthe observed decrease in ethylene production was not due to inactivationof the efe gene.

Higher EFE protein levels were also observed with the expression of efefrom the psbA promoter compared to the petE promoter. The increase inexpression from the psbA promoter was corroborated by a 10-fold higherethylene production from this strain (see FIG. 16A). The psbA:EFE lineswere then examined to determine if ethylene production could be stablymaintained over several generations in this strain. In order to examinestability of this ethylene producing strain, the cultures were seriallypassed through 4 generations of growth and ethylene production wasmeasured daily for 10 days with each generation. In contrast to previousstudies in which the ability of a culture to produce ethylene decreasedover successive generations and had completely disappeared after threegenerations (Takahama et al., J. Biosci. Bioeng. 95:302-305 (2003)),consistent rates of ethylene production were maintained through thecourse of 4 serial passages of the same culture (see FIG. 16B). Thissuggests that the Synechocystis strain was capable of maintaining afunctional copy of the modified efe gene over multiple serial passagesof the culture.

Example 9 Effect of Medium on Ethylene Production

Ethylene production peaks early in the culture's growth period andtypically falls off as the culture ages (see FIG. 16B). Ethyleneproduction rates were restored each time in successive subculture,indicating that the decrease in ethylene production was not due toinactivation of efe. One possible reason for the apparent decrease inethylene production is that light, carbon, or another nutrient becomeslimiting as the culture reaches stationary phase. In order to elucidatethe limiting component(s) for ethylene production, a stationary phaseculture in which ethylene production had nearly ceased was divided andtransferred to cultures with high light (150 μm⁻²s) exposure, addedadditional nitrogen, or fresh growth medium.

The culture that was supplemented with additional ammonium displayed arepressed ethylene production rate compared to the control (see FIG.17A). The culture that was transferred to high light reached asignificantly higher density. However, this culture produced a similaramount of ethylene as the control despite the fact that there were 1.5times as many cells in the culture. In contrast, the culture that wasresuspended in fresh medium reached a similar density as the control yetproduced more ethylene. Collectively, these findings suggest thatmaximum cell density and ethylene production are limited by independentfactors, with light limiting the final cell density and component(s) ofthe medium limiting ethylene production.

To determine whether increasing the medium concentration could furtherstimulate ethylene production, medium concentrations ranging between 1×and 10× were tested. Increasing the medium concentration as much as10-fold indeed promoted increasing rates of ethylene production (seeFIG. 17B). 10× medium led to a 2-fold increase in total ethyleneproduction relative to 1× medium, producing a peak rate of 700 nL mL⁻¹hr⁻¹.

The ability of medium replenishment to restore ethylene production froma culture that had ceased to produce ethylene was also investigated. Astationary phase culture that had lost its ability to produce ethylenewas resuspended in fresh 5× medium at the beginning of each week for amonth. Ethylene production was measured each day over the course of theexperiment. Ethylene production from a small sample of the culture wasalso measured before it was resuspended to confirm that the culture hadceased to produce high levels of ethylene prior to resuspension.High-level ethylene production was determined to resume immediatelyafter resuspension in fresh medium; reaching a peak three days later andthen declining thereafter. Additionally, each time the medium wasrefreshed, the peak production rate for that culture also increased,eventually reaching a maximum of 1500 nL mL⁻¹ hr⁻¹ (see FIG. 17C). Thesefindings suggest that a culture will maintain high-level ethyleneproduction provided the problem of medium limitation is alleviated.

Example 10 Expression of Multiple Copies of EFE

Because expression of EFE produced no apparent metabolic burden onSynechocystis, ethylene production in strains expressing a second copyof efe was investigated. The second copy of efe was also under controlof the pea plant psbA promoter, and was integrated into theSynechocystis genome at the psbA2 locus along with a kanamycinresistance cassette. Increased expression of EFE was verified by Westernblotting. Comparison of ethylene production between strains with asingle or double copy of efe showed that a second copy of efe doubledthe ethylene output (FIG. 18). Furthermore, the strain with a secondcopy of efe exhibited growth characteristics similar to wild type (Table2), suggesting that increased ethylene production does not pose a severemetabolic burden.

TABLE 2 Strain Generation time (h) wild type 10.7 +/− 0.41 psbA:Sy-efe11.0 +/− 0.58 2X psbA:Sy-efe 10.9 +/− 0.63

Example 11 Ethylene Production in Semi-continuous Culture

Since depletion of medium components limits ethylene productivity,regular medium replenishment should sustain high level production. Totest this, ethylene production in semi-continuous culture expressingeither one or two copies of efe was examined. A stationary-phase culturewas diluted to an OD₇₃₀ of 0.25, allowing it to resume log phase growth.Seven days later, the culture reached stationary phase and ceasedproducing significant amounts of ethylene. The culture was then spundown and resuspended at the same cell concentration in fresh 5×BG11. Theprocess of resuspending the previous culture (without dilution) in fresh5×BG11 was repeated weekly for 3 additional weeks. Ethylene productionwas measured each day over the course of the experiment. High-levelethylene production resumed immediately after resuspension in freshmedium, reaching a peak 24 hours later and declined thereafter (FIG.19). Each time the medium was refreshed, growth resumed, and the peakproduction rate for that culture also increased, eventually reaching amaximum of 3100 μL L⁻¹ h⁻¹ at OD₇₃₀ of approximately 20. Specificactivity generally decreased as the culture density increased, but wasmore than offset by the increase in culture density, resulting in highertotal production from high density cultures.

Weekly media replacement was insufficient to sustain ethylene productionat the peak level (3100 μL L⁻¹ h⁻¹) for longer than one day; thusconditions more closely resembling continuous culture were investigatedto determine if this would extend ethylene production at the maximumrate. Daily media refreshment was performed to maintain a specificculture density. A culture expressing two copies of efe was concentratedto an initial density of OD₇₃₀ 15.0. Each day for three weeks theculture was spun down and resuspended at this same density; ethyleneproduction was measured after each resuspension. Under these conditionsthe peak production rate of 3100 μL L⁻¹ h⁻¹ was maintained continuously(FIG. 19). This finding indicates that the peak rates reported in thisstudy resemble the continuous rate that can be expected using continuousculture.

Example 12 Effect of Light Intensity on Ethylene Production

Increases in total productivity can be achieved by using cultures ofincreasing density. The potential to obtain a similar increase inproduction on a per cell basis was also investigated. Growing cells atvery high density can result in light limitation due to self shading, soalleviating this limitation could increase the specific productivity.The effect of various light intensities, up to 350 μE, on specificethylene production was examined. The results presented in FIG. 20Bdemonstrate that specific productivity increases with increasing lightintensity. With high light intensity, a measured total productivity of2500 mL mL⁻¹ hr⁻¹ was achieved (see FIG. 20A). This production rateresulted from a combination of increased specific productivity whilemaintaining the high culture density that previously yielded aproduction rate of 1500 mL mL⁻¹ hr⁻¹, rather than just using more cellsto generate increasing ethylene production rates.

The effect of high light (600 μE m⁻²s⁻¹) on ethylene production rates ofhigh cell density (OD⁷³⁰ ˜15.0) was also examined. Cultures expressingeither one or two copies of efe were resuspended in fresh 5×BG11 to adensity of OD₇₃₀ approximately 15 and placed under all white Diamondseries LED lights (Advanced LED Lights) with 600 μE m⁻²s⁻¹ reaching theculture. Ethylene production was measured daily for a week. As shown inFIG. 21, high light (600 μE m⁻²s⁻¹) approximately doubled the ethyleneproduction from both strains compared to 50 μE m⁻²s⁻¹, with the peakrate reaching 5700 μL L⁻¹ h⁻¹ for the strain expressing two copies ofefe. The culture growing in high light also reached a higher densitythan the one grown in low light.

Example 13 Ethylene Production in Sea Water

Competition for fresh water and arable land is a serious concern forbiofuel production. To determine whether sea water could serve as asubstitute for 5×BG11, ethylene production in the presence of regularseawater, 5×BG11 media, or 5×BG11 medium made with sea water wasexamined. Filter sterilized sea-pure (Caribsea) ocean water was used tosubstitute for BG11 in sea-water experiments. Ethylene production wasimpaired in sea water; however, seawater supplemented with 4 mg/Lphosphate and 150 mg/L nitrate (the same concentrations that are used in5×BG11) could support growth and ethylene production at rates comparableto those observed with 5×BG11 medium (FIG. 24). Replacing growth mediumwith supplemented sea water can lower the production cost of ethyleneand conserve fresh water for other uses. Furthermore, photosyntheticethylene production could be located in brackish water bodies, sea bayor coastal areas where it would not compete for arable land and freshwater.

The Examples discussed above are provided for purposes of illustrationand are not intended to be limiting. Still other embodiments andmodifications are also contemplated.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

We claim:
 1. A method for producing alpha ketoglutarate (AKG) orpyruvate, comprising: a) culturing a cyanobacterial cell that lacks afunctional ADP-glucose pyrophosphorylase (AGP) enzyme under conditionsthat allow for AKG or pyruvate production, and b) recovering the AKG orpyruvate from the cyanobacterial cell culture.
 2. The method of claim 1,wherein the cyanobacterial cell does not express a functional glgC gene.3. The method of claim 1, wherein the cyanobacterial cell is aSynechocystis cell.
 4. The method of claim 1, wherein the cyanobacterialcell is a Synechocystis sp. PCC 6803 cell.
 5. The method of claim 1,wherein the cyanobacterial cell is cultured in media that does notcontain nitrogen.
 6. The method of claim 5, wherein the concentration ofnitrogen in the media is less than about 200 μM.
 7. The method of claim6, further comprising a step of adding nitrogen to the media at a finalconcentration of less than about 1 mM.
 8. The method of claim 6, whereinthe cyanobacterial cell is cultured under a light intensity of at leastabout 350 μE m⁻²s⁻¹.
 9. The method of claim 6, wherein thecyanobacterial cell is cultured under a light intensity of at leastabout 600 μE m⁻²s⁻¹.
 10. The method of claim 1, wherein the AKGconcentration in the culture is greater than 100 mg per liter.
 11. Themethod of claim 1, wherein the AKG concentration in the culture isgreater than 1000 mg per liter.
 12. The method of claim 1, wherein thecyanobacterial cell exhibits at least a 10,000-fold increase in AKGproduction when compared to a wild type cell.
 13. The method of claim 1,wherein the pyruvate concentration in the culture is greater than 1 gper liter.
 14. The method of claim 1, wherein the pyruvate concentrationin the culture is greater than 100 g per liter.
 15. The method of claim1, wherein the cyanobacterial cell exhibits at least a 10,000-foldincrease in pyruvate production when compared to a wild type cell.
 16. Acyanobacterial cell that lacks a functional ADP-glucosepyrophosphorylase (AGP) enzyme and produces 10,000-fold more AKG orpyruvate when compared to a wild type cell.
 17. The cyanobacterial cellof claim 16, wherein the cyanobacterial cell does not express afunctional glgC gene.
 18. The cyanobacterial cell of claim 16, whereinthe cyanobacterial cell is a Synechocystis cell.
 19. The cyanobacterialcell of claim 16, wherein the cyanobacterial cell is a Synechocystis sp.PCC 6803 cell.